Antisense modulation of phosphorylase kinase alpha 1 expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of Phosphorylase kinase alpha 1. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding Phosphorylase kinase alpha 1. Methods of using these compounds for modulation of Phosphorylase kinase alpha 1 expression and for treatment of diseases associated with expression of Phosphorylase kinase alpha 1 are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of Phosphorylase kinase alpha 1. In particular, thisinvention relates to compounds, particularly oligonucleotides,specifically hybridizable with nucleic acids encoding Phosphorylasekinase alpha 1. Such compounds have been shown to modulate theexpression of Phosphorylase kinase alpha 1.

BACKGROUND OF THE INVENTION

Balanced energy metabolism is critical to the regulation of allbiological processes. In higher organisms, energy stores are in the formof glycogen. Upon energy deficit, these stores are mobilized throughenzymatic digestion to glucose-1-phosphate by a diverse set of signalsand are used to maintain blood-glucose levels, as a source of energyduring muscle contraction and as source of fuel for a broad range ofcellular activities.

The protein kinase, phosphorylase kinase (PHK) plays a central role inthe regulation of glycogen degradation or glycogenolysis byphosphorylating glycogen phosphorylase b, a unique reaction catalyzedonly by phosphorylase kinase. It also lies at the interface betweensignaling and metabolic pathways and translates the pleiotropic actionsof extracellular signals, including hormonal and neuronal, into specificand directional intracellular responses. In addition, phosphorylasekinase can express varying degrees of activity depending on pH, metalion concentration, allosteric effectors and covalent modifications(Brushia and Walsh, Front. Biosci., 1999, 4, D618-641).

Structurally, phosphorylase kinase is one of the most complex enzymesisolated to date, a hexadecamer, having three distinct regulatorysubunits, alpha, beta and delta (also known as calmodulin), and onecatalytic subunit, gamma. Each holoenzyme is composed of fourheterotetramers of the component subunits and the subunit stoichiometryhas been shown to vary depending on the tissue source. The phosphorylasekinase subunits also exist as multiple isoforms adding an additionallayer of complexity. The alpha, beta, and gamma isoforms are foundexpressed in the liver and muscle with minor amounts in the gut, whilethe delta (calmodulin) isoforms are expressed in all tissues examined(Brushia and Walsh, Front. Biosci., 1999, 4, D618-641).

Due to the direct relationship between phosphorylase kinase enzymeactivity and maintenance of blood-glucose homeostasis, modifications tothe regulatory properties of this enzyme could provide great therapeuticbenefit in the arena of metabolic disorders, especially diabetes.

Phosphorylase kinase alpha 1 (also known as PHKA, PHKA1 and αM) is oneof the three regulatory alpha subunit isoforms identified to date and islocalized solely in muscle tissue (Wullrich et al., J. Biol. Chem.,1993, 268, 23208-23214).

The gene is located on chromosome Xq13 and several types of mutations inthis gene have been reported which result in differential mRNAprocessing and certain forms of glycogen storage diseases. Thesemutations include a splice junction mutation in a patient with myopathy(Bruno et al., Biochem. Biophys. Res. Commun., 1998, 249, 648-651) andnonsense mutations that resemble the X-linked phosphorylase kinasedeficiency seen in I-strain mice (Wehner et al., Hum. Mol. Genet., 1994,3, 1983-1987). In mice this nonsense mutation results in a frameshift ofthe coding region and therefore disrupts the expression of both theliver and muscle isoforms of the alpha subunit (Bender, Biochem.Biophys. Res. Commun., 1991, 179, 707-712; Bender and Lalley, Proc.Natl. Acad. Sci. U.S.A., 1989, 86, 9996-10000; Schneider et al., Nat.Genet., 1993, 5, 381-385).

Currently however, there are no known therapeutic agents whicheffectively inhibit the synthesis of phosphorylase kinase alpha 1 and todate, investigative strategies aimed at studying phosphorylase kinasealpha 1 function have involved the use of antibodies and crosslinkingagents.

Consequently, there remains a long felt need for agents capable ofeffectively modulating phosphorylase kinase alpha 1 function.

Antisense technology is emerging as an effective means for reducing theexpression of specific gene products and may therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications for the modulation of phosphorylase kinase alpha 1expression.

The present invention provides compositions and methods for modulatingphosphorylase kinase alpha 1 expression.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, particularly antisenseoligonucleotides, which are targeted to a nucleic acid encodingPhosphorylase kinase alpha 1, and which modulate the expression ofPhosphorylase kinase alpha 1. Pharmaceutical and other compositionscomprising the compounds of the invention are also provided. Furtherprovided are methods of modulating the expression of Phosphorylasekinase alpha 1 in cells or tissues comprising contacting said cells ortissues with one or more of the antisense compounds or compositions ofthe invention. Further provided are methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of Phosphorylase kinase alpha 1 byadministering a therapeutically or prophylactically effective amount ofone or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding Phosphorylase kinase alpha 1, ultimatelymodulating the amount of Phosphorylase kinase alpha 1 produced. This isaccomplished by providing antisense compounds which specificallyhybridize with one or more nucleic acids encoding Phosphorylase kinasealpha 1. As used herein, the terms “target nucleic acid” and “nucleicacid encoding Phosphorylase kinase alpha 1” encompass DNA encodingPhosphorylase kinase alpha 1, RNA (including pre-mRNA and mRNA)transcribed from such DNA, and also cDNA derived from such RNA. Thespecific hybridization of an oligomeric compound with its target nucleicacid interferes with the normal function of the nucleic acid. Thismodulation of function of a target nucleic acid by compounds whichspecifically hybridize to it is generally referred to as “antisense”.The functions of DNA to be interfered with include replication andtranscription. The functions of RNA to be interfered with include allvital functions such as, for example, translocation of the RNA to thesite of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity which may be engaged in or facilitated by the RNA. The overalleffect of such interference with target nucleic acid function ismodulation of the expression of Phosphorylase kinase alpha 1. In thecontext of the present invention, “modulation” means either an increase(stimulation) or a decrease (inhibition) in the expression of a gene. Inthe context of the present invention, inhibition is the preferred formof modulation of gene expression and mRNA is a preferred target.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding Phosphorylase kinase alpha 1. The targeting processalso includes determination of a site or sites within this gene for theantisense interaction to occur such that the desired effect, e.g.,detection or modulation of expression of the protein, will result.Within the context of the present invention, a preferred intragenic siteis the region encompassing the translation initiation or terminationcodon of the open reading frame (ORF) of the gene. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon”. A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding Phosphorylase kinase alpha 1, regardless of thesequence(s) of such codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5′ untranslatedregion (5′UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense and other compounds of the invention which hybridize to thetarget and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds which hybridize to these active sites.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat oligonucleotides can be useful therapeutic modalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. This term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentinternucleoside (backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 50 nucleobases (i.e.from about 8 to about 50 linked nucleosides). Particularly preferredantisense compounds are antisense oligonucleotides, even more preferablythose comprising from about 12 to about 30 nucleobases. Antisensecompounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be abasic (the nucleobase is missing or has a hydroxylgroup in place thereof). Various salts, mixed salts and free acid formsare also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, aO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃, alsoknown as 2′—O—(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′—O—dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′—O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

A further prefered modification includes Locked Nucleic Acids (LNAs) inwhich the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of thesugar ring thereby forming a bicyclic sugar moiety. The linkage ispreferably a methelyne (—CH₂—). group bridging the 2′ oxygen atom andthe 3′ or 4′ carbon atom wherein n is 1 or 2. LNAs and preparationthereof are described in WO 98/39352 and WO 99/14226.

Other preferred modifications include 2′-methoxy (2′—O—CH₃),2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂), 2′-allyl (2′—CH₂—CH═CH₂),2′—O—allkyl (2′—O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modificationmay be in the arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. The compounds of the invention caninclude conjugate groups covalently bound to functional groups such asprimary or secondary hydroxyl groups. Conjugate groups of the inventioninclude intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of Phosphorylase kinase alpha 1 is treated by administeringantisense compounds in accordance with this invention. The compounds ofthe invention can be utilized in pharmaceutical compositions by addingan effective amount of an antisense compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the antisensecompounds and methods of the invention may also be usefulprophylactically, e.g., to prevent or delay infection, inflammation ortumor formation, for example.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingPhosphorylase kinase alpha 1, enabling sandwich and other assays toeasily be constructed to exploit this fact. Hybridization of theantisense oligonucleotides of the invention with a nucleic acid encodingPhosphorylase kinase alpha 1 can be detected by means known in the art.Such means may include conjugation of an enzyme to the oligonucleotide,radiolabelling of the oligonucleotide or any other suitable detectionmeans. Kits using such detection means for detecting the level ofPhosphorylase kinase alpha 1 in a sample may also be prepared.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Preferredlipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidylglycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively,oligonucleotides may be complexed to lipids, in particular to cationiclipids. Preferred fatty acids and esters include but are not limitedarachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylicacid, capric acid, myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine,an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM),monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.Topical formulations are described in detail in U.S. patent applicationSer. No. 09/315,298 filed on May 20, 1999 which is incorporated hereinby reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Prefered bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,. Preferedfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also prefered are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly prefered combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed July 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999) each of which is incorporated herein by referencein their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other. In general, emulsions may be eitherwater-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueousphase is finely divided into and dispersed as minute droplets into abulk oily phase the resulting composition is called a water-in-oil (w/o)emulsion. Alternatively, when an oily phase is finely divided into anddispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil-in-water (o/w) emulsion. Emulsions maycontain additional components in addition to the dispersed phases andthe active drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtriglycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1 ndWO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p.92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited todaunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Numerous examples of antisensecompounds are known in the art. Two or more combined compounds may beused together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-Alkoxy Amidites

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham Mass. or GlenResearch, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleosideamidites are prepared as described in U.S. Pat. 5,506,351, hereinincorporated by reference. For oligonucleotides synthesized using2′-alkoxy amidites, the standard cycle for unmodified oligonucleotideswas utilized, except the wait step after pulse delivery of tetrazole andbase was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2′-Fluoro Amidites

2′-Fluorodeoxyadenosine Amidites

2′-fluoro oligonucleotides were synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyrylarabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′ phosphoramidites.

2′-Fluorodeoxycytidine

2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-O-(2-Methoxyethyl) Modified Amidites

2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.).

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by TLC by first quenching the TLC sample with the addition ofMeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH3 gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2,-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, TLC showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(Dimethylaminooxyethyl) Nucleoside Amidites

2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the artas 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L)and brine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to −10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 mL) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2kg silica gel, ethyl acetate-hexanesgradient 1:1 to 4:1). The appropriate fractions were combined, strippedand dried to product as a white crisp foam (84 g, 50%), contaminatedstarting material (17.4 g) and pure reusable starting material 20 g. Theyield based on starting material less pure recovered starting materialwas 58%. TLC and NMR were consistent with 99% pure product.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0C.After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂C1₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂C1₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P205 under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl2 (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂0₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-31-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) Nucleoside Amidites

2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-Isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog may be obtained by selective2′-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minoramount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine ribosidemay be resolved and converted to 2′-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection proceduresshould afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosineand2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the artas 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleosideamidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

2-[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowlyadded to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methylUridine

To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂C₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

5′-O-Dimnethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2 Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3 Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[21-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphor-amidite for the DNA portionand 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′wings. The standard synthesis cycle is modified by increasing the waitstep after the delivery of tetrazole and base to 600 s repeated fourtimes for RNA and twice for 2′-O-methyl. The fully protectedoligonucleotide is cleaved from the support and the phosphate group isdeprotected in 3:1 ammonia/ethanol at room temperature overnight thenlyophilized to dryness. Treatment in methanolic ammonia for 24 hrs atroom temperature is then done to deprotect all bases and sample wasagain lyophilized to dryness. The pellet is resuspended in 1M TBAF inTHF for 24 hrs at room temperature to deprotect the 2′ positions. Thereaction is then quenched with 1M TEAA and the sample is then reduced to½ volume by rotovac before being desalted on a G25 size exclusioncolumn. The oligo recovered is then analyzed spectrophotometrically foryield and for purity by capillary electrophoresis and by massspectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toUnited States patent 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing 6 cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, Ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

NHDF cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

3T3-L1 Cells:

The mouse embryonic adipocyte-like cell line 3T3-Ll was obtained fromthe American Type Culure Collection (Manassas, Va.). 3T3-Ll cells wereroutinely cultured in DMEM, high glucose (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 80% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 4000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A10 Cells:

The rat aortic smooth muscle cell line A10 was obtained from theAmerican Type Culure Collection (Manassas, Va.). A10 cells wereroutinely cultured in DMEM, high glucose (American Type CulureCollection, Manassas, Va.) supplemented with 10% fetal calf serum(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 80%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 2500 cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Treatment with Antisense Compounds:

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEMm-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10 Analysis of Oligonucleotide Inhibition of PhosphorylaseKinase Alpha 1 Expression

Antisense modulation of Phosphorylase kinase alpha 1 expression can beassayed in a variety of ways known in the art. For example,Phosphorylase kinase alpha 1 mRNA levels can be quantitated by, e.g.,Northern blot analysis, competitive polymerase chain reaction (PCR), orreal-time PCR (RT-PCR). Real-time quantitative PCR is presentlypreferred. RNA analysis can be performed on total cellular RNA orpoly(A)+ mRNA. Methods of RNA isolation are taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northernblot analysis is routine in the art and is taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative(PCR) can be conveniently accomplished using the commercially availableABI PRISfM™ 7700 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Protein levels of Phosphorylase kinase alpha 1 can be quantitated in avariety of ways well known in the art, such as immunoprecipitation,Western blot analysis (immunoblotting), ELISA or fluorescence-activatedcell sorting (FACS). Antibodies directed to Phosphorylase kinase alpha 1can be identified and obtained from a variety of sources, such as theMSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), orcan be prepared via conventional antibody generation methods. Methodsfor preparation of polyclonal antisera are taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2,pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation ofmonoclonal antibodies is taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5,John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11 Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 FL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC™ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-time Quantitative PCR Analysis of Phosphorylase KinaseAlpha 1 mRNA Levels

Quantitation of Phosphorylase kinase alpha 1 mRNA levels was determinedby real-time quantitative PCR using the ABI PRISM™ 7700 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR, in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., JOE, FAM, or VIC, obtained from either OperonTechnologies Inc., Alameda, Calif. or PE-Applied Biosystems, FosterCity, Calif.) is attached to the 5′ end of the probe and a quencher dye(e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda,Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the3′ end of the probe. When the probe and dyes are intact, reporter dyeemission is quenched by the proximity of the 3′ quencher dye. Duringamplification, annealing of the probe to the target sequence creates asubstrate that can be cleaved by the 5′-exonuclease activity of Taqpolymerase. During the extension phase of the PCR amplification cycle,cleavage of the probe by Taq polymerase releases the reporter dye fromthe remainder of the probe (and hence from the quencher moiety) and asequence-specific fluorescent signal is generated. With each cycle,additional reporter dye molecules are cleaved from their respectiveprobes, and the fluorescence intensity is monitored at regular intervalsby laser optics built into the ABI PRISM™ 7700 Sequence DetectionSystem. In each assay, a series of parallel reactions containing serialdilutions of mRNA from untreated control samples generates a standardcurve that is used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1×TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLtotal RNA solution. The RT reaction was carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent from Molecular Probes. Methods of RNA quantification byRiboGreen™ are taught in Jones, L. J., et al, Analytical Biochemistry,1998, 265, 368-374.

In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 25 uL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nmand emission at 520 nm.

Probes and primers to human Phosphorylase kinase alpha 1 were designedto hybridize to a human Phosphorylase kinase alpha 1 sequence, usingpublished sequence information (GenBank accession number X73874,incorporated herein as SEQ ID NO:3). For human Phosphorylase kinasealpha 1 the PCR primers were:

forward primer: GACTTCGGGATATGGGAACGT (SEQ ID NO: 4)

reverse primer: TCACACCAAACAGATCCAGTTCA (SEQ ID NO: 5) and the PCR probewas: FAM-AACCAAGGGATCTCAGAGTTGAATGCCA-TAMRA (SEQ ID NO: 6) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye. For human GAPDH the PCR primers were:

forward primer: CAACGGATTTGGTCGTATTGG (SEQ ID NO: 7)

reverse primer: GGCAACAATATCCACTTTACCAGAGT (SEQ ID NO: 8) and the PCRprobe was: 5′JOE-CGCCTGGTCACCAGGGCTGCT-TAMRA 3′ (SEQ ID NO: 9) where JOE(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye.

Probes and primers to mouse Phosphorylase kinase alpha 1 were designedto hybridize to a mouse Phosphorylase kinase alpha 1 sequence, usingpublished sequence information (GenBank accession number X74616,incorporated herein as SEQ ID NO:10). For mouse Phosphorylase kinasealpha 1 the PCR primers were:

forward primer: GGGAACGTGGCGATAAGACA (SEQ ID NO:11)

reverse primer: ACCAAACAAGTCTAATTCATCTAGTGCTT (SEQ ID NO: 12) and thePCR probe was: FAM-CCAAGGCATCTCGGAATTGAATGCG-TAMRA (SEQ ID NO: 13) whereFAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescentreporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) isthe quencher dye. For mouse GAPDH the PCR primers were:

forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 14)

reverse primer: GGGTCTCGCTCCTGGAAGCT (SEQ ID NO: 15) and the PCR probewas: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 16) whereJOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescentreporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) isthe quencher dye.

Probes and primers to rat Phosphorylase kinase alpha 1 were designed tohybridize to a rat Phosphorylase kinase alpha 1 sequence, usingpublished sequence information (GenBank accession number M92918,incorporated herein as SEQ ID NO:17). For rat Phosphorylase kinase alpha1 the PCR primers were:

forward primer: TGAAGATGGAGCCAGCTTGA (SEQ ID NO: 18)

reverse primer: CACCAAAATAACCATCCTGCATT (SEQ ID NO: 19) and the PCRprobe was: FAM-TCAAGTATCCTGGCAGCACTCCGGA-TAMRA (SEQ ID NO: 20) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye. For ratO GAPDH the PCR primers were:

forward primer: TGTTCTAGAGACAGCCGCATCTT (SEQ ID NO: 21)

reverse primer: CACCGACCTTCACCATCTTGT (SEQ ID NO: 22) and the PCR probewas: 5′ JOE-TTGTGCAGTGCCAGCCTCGTCTCA-TAMRA 3′ (SEQ ID NO: 23) where JOE(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye.

Example 14 Northern Blot Analysis of Phosphorylase Kinase Alpha 1 mRNALevels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human Phosphorylase kinase alpha 1, a human Phosphorylasekinase alpha 1 specific probe was prepared by PCR using the forwardprimer GACTTCGGGATATGGGAACGT (SEQ ID NO: 4) and the reverse primerTCACACCAAACAGATCCAGTTCA (SEQ ID NO: 5). To normalize for variations inloading and transfer efficiency membranes were stripped and probed forhuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

To detect mouse Phosphorylase kinase alpha 1, a mouse Phosphorylasekinase alpha 1 specific probe was prepared by PCR using the forwardprimer GGGAACGTGGCGATAAGACA (SEQ ID NO:11) and the reverse primerACCAAACAAGTCTAATTCATCTAGTGCTT (SEQ ID NO: 12). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

To detect rat Phosphorylase kinase alpha 1, a rat Phosphorylase kinasealpha 1 specific probe was prepared by PCR using the forward primerTGAAGATGGAGCCAGCTTGA (SEQ ID NO: 18) and the reverse primerCACCAAAATAACCATCCTGCATT (SEQ ID NO: 19). To normalize for variations inloading and transfer efficiency membranes were stripped and probed forrat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, PaloAlto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Inhibition of Human Phosphorylase Kinase Alpha 1Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-NOEWings and a Deoxy Gap

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human Phosphorylasekinase alpha 1 RNA, using published sequences (GenBank accession numberX73874, incorporated herein as SEQ ID NO: 3, and GenBank accessionnumber X73878, incorporated herein as SEQ ID NO: 24). Theoligonucleotides are shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humanPhosphorylase kinase alpha 1 mRNA levels by quantitative real-time PCRas described in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”.

TABLE 1 Inhibition of human Phosphorylase kinase alpha 1 mRNA levels bychimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap ISIS TARGET TARGET SEQ ID # REGION SEQ ID NO SITE SEQUENCE %INHIB NO 118465 Start 3 153 cggctcctcatggcgacacg 77 25 Codon 118466Coding 3 219 tgatggcacaggatggtctg 71 26 118467 Coding 3 252tagctggctggaagcaagcc 76 27 118468 Coding 3 291 ctgtacacattatctcggac 9028 118469 Coding 3 316 gcccaaaccccacacagcca 98 29 118470 Coding 3 391cttcactacactctgctcca 89 30 118471 Coding 3 426 tgtctgatcatgcagtgcag 9331 118472 Coding 3 438 actttatccacctgtctgat 91 32 118473 Coding 3 549tccaactgcaggtgtcccca 99 33 118474 Coding 3 600 agtcctgaggcagtcatttg 9934 118475 Coding 3 620 ctaggctgtggatgatatgg 86 35 118476 Coding 3 675gttttatatgcagcttcaat 50 36 118477 Coding 3 811 cccacctttcacaccaaaca 9037 118478 Coding 3 834 aggacatggataactgattg 88 38 118479 Coding 3 1008ccctgaagcttggtgatgat 85 39 118480 Coding 3 1019 aaccataacgaccctgaagc 8940 118481 Coding 3 1056 ttaggagttttatatccatc 95 41 118482 Coding 3 1104aatagcttcagctcagctgg 81 42 118483 Coding 3 1140 gtccagaacaatggccattc 9843 118484 Coding 3 1196 tatattcttgaacctgttct 95 44 118485 Coding 3 1230ttgcccttgatgaggactgc 72 45 118486 Coding 3 1350 ccccacatgtgaggcaattt 9246 118487 Coding 3 1360 tagagactgaccccacatgt 94 47 118488 Coding 3 1390tccctctgccatcaagcttc 73 48 118489 Coding 3 1476 agaatggagacttgaaccac 8549 118490 Coding 3 1497 ttgatttcttctgtttcagc 96 50 118491 Coding 3 1584tggctgagaatacgagctgg 94 51 118492 Coding 3 1631 gtccactgagtttcattcta 8852 118493 Coding 3 1704 gtgaaagtaaagatagtttt 39 53 118494 Coding 3 1714aaactgtggagtgaaagtaa 73 54 118495 Coding 3 1734 tagaactgttgctggtctat 8155 118496 Coding 3 1746 tccagagccaggtagaactg 97 56 118497 Coding 3 1803cagcggctacagaggtagga 82 57 118498 Coding 3 1825 ggtgggctggcctgtcatcc 9158 118499 Coding 3 1868 ttccatcttcatcaagcatg 99 59 118500 Coding 3 1890aggatacttgaattcaagct 71 60 118501 Coding 3 1909 catttttcggagtgctgcca 8561 118502 Coding 3 1959 tctgacaatttacctgtttg 99 62 118503 Coding 3 1972tgttgtcaaaaactctgaca 76 63 118504 Coding 3 1977 caagatgttgtcaaaaactc 8764 118505 Coding 3 2010 ggtccagggtccatgaagct 73 65 118506 Coding 3 2021gcttaccctcaggtccaggg 96 66 118507 Coding 3 2041 atcataatcttcactgtaca 7667 118508 Coding 3 2119 ttcatcaccacagcgagcat 80 68 118509 Coding 3 2207accgatctagccctcccttc 94 69 118510 Coding 3 2250 accaaggacattaagtcgca 9670 118511 Coding 3 2293 atacatgtgaacattctgta 88 71 118512 Coding 3 2465ttgaggtctccttcaactgt 94 72 118513 Coding 3 2526 ttccagtcaggtcctttcat 9173 118514 Coding 3 2676 gtgcaggcctcatcaagtgc 30 74 118515 Coding 3 2722tggaggaagtcctactgtca 97 75 118516 Coding 3 2794 actggcttcatctatcagct 9576 118517 Coding 3 2837 ccattatttcctgtgtaagg 92 77 118518 Coding 3 2926tgccataacttgtatgatca 86 78 118519 Coding 3 2973 tctgtggcttcctcagctga 5779 118520 Coding 3 2993 gactgagattcatcaggccc 93 80 118521 Coding 3 3030ctgagaatgtgatgcaggag 77 81 118522 Coding 3 3057 cttcgttccactccaaactc 6382 118523 Coding 3 3077 ttgaatcagtgggacgaacg 74 83 118524 Coding 3 3123gctccaacagcaccaatctc 94 84 118525 Coding 3 3165 tcactttttaactgcatgat 8085 118526 Coding 3 3185 gaaattccacctgctttatc 17 86 118527 Coding 3 3295ttgacgactatctttagatg 67 87 118528 Coding 3 3465 gagaatttaatctcacctgg 4988 118529 Coding 3 3489 ttcaggacagactccacatg 87 89 118530 Coding 3 3540acaaggatggcttcaaccag 93 90 118531 Coding 3 3561 atatctgccagcatggtgag 8691 118532 Coding 3 3571 atgaatttcaatatctgcca 92 92 118533 Coding 3 3639tgttcttgaaggaacaagtc 67 93 118534 Coding 3 3669 aacatggtatcatctgcgcc 9694 118535 Coding 3 3679 atcctttgccaacatggtat 75 95 118536 Coding 3 3702agagtacagatgccagatgc 92 96 118537 Coding 3 3727 gccactgggtgcactgtcat 7397 118538 Coding 3 3750 aggtaggtcatggtgccaaa 92 98 118539 Coding 3 3783aactcctgcacgtaggtggc 85 99 118540 Stop 3 3823 ccaaagccctcattgcatgg 76100 Codon 118541 3′UTR 24 102 gtccaagctatgacaagcta 41 101 118542 3′UTR24 351 ataatgggtattattaaaag 15 102

As shown in Table 1, SEQ ID NOs 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 87, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99 and 100 demonstrated at least 60% inhibition of humanPhosphorylase kinase alpha 1 expression in this assay and are thereforepreferred. The target sites to which these preferred sequences arecomplementary are herein referred to as “active sites” and are thereforepreferred sites for targeting by compounds of the present invention.

Example 16 Antisense Inhibition of Mouse Phosphorylase Kinase Alpha 1Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the mousePhosphorylase kinase alpha 1 RNA, using published sequences (GenBankaccession number X74616, incorporated herein as SEQ ID NO: 10). Theoligonucleotides are shown in Table 2. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 2 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-nucleotides, which is flankedon both sides (5′ and 3′ directions) by five-nucleotide “wings”. Thewings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mousePhosphorylase kinase alpha 1 mRNA levels by quantitative real-time PCRas described in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”.

TABLE 2 Inhibition of mouse Phosphorylase kinase alpha 1 mRNA levels bychimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap ISIS TARGET TARGET SEQ ID # REGION SEQ ID NO SITE SEQUENCE %INHIB NO 118466 Coding 10 276 tgatggcacaggatggtctg 47 26 118467 Coding10 309 tagctggctggaagcaagcc 49 27 118468 Coding 10 348ctgtacacattatctcggac 63 28 118469 Coding 10 373 gcccaaaccccacacagcca 7129 118470 Coding 10 448 cttcactacactctgctcca 40 30 118471 Coding 10 483tgtctgatcatgcagtgcag 52 31 118472 Coding 10 495 actttatccacctgtctgat 6932 118473 Coding 10 606 tccaactgcaggtgtcccca 82 33 118474 Coding 10 657agtcctgaggcagtcatttg 62 34 118475 Coding 10 677 ctaggctgtggatgatatgg 2535 118476 Coding 10 732 gttttatatgcagcttcaat 27 36 118477 Coding 10 868cccacctttcacaccaaaca 63 37 118478 Coding 10 891 aggacatggataactgattg 5238 118479 Coding 10 1065 ccctgaagcttggtgatgat 66 39 118480 Coding 101076 aaccataacgaccctgaagc 22 40 118481 Coding 10 1113ttaggagttttatatccatc 37 41 118482 Coding 10 1161 aatagcttcagctcagctgg 5242 118483 Coding 10 1197 gtccagaacaatggccattc 73 43 118484 Coding 101253 tatattcttgaacctgttct 67 44 118485 Coding 10 1287ttgcccttgatgaggactgc 23 45 118486 Coding 10 1407 ccccacatgtgaggcaattt 4246 118487 Coding 10 1417 tagagactgaccccacatgt 29 47 118488 Coding 101447 tccctctgccatcaagcttc 24 48 118489 Coding 10 1533agaatggagacttgaaccac 41 49 118490 Coding 10 1554 ttgatttcttctgtttcagc 4350 118491 Coding 10 1641 tggctgagaatacgagctgg 20 51 118492 Coding 101688 gtccactgagtttcattcta 48 52 118493 Coding 10 1761gtgaaagtaaagatagtttt 0 53 118494 Coding 10 1771 aaactgtggagtgaaagtaa 4254 118495 Coding 10 1791 tagaactgttgctggtctat 46 55 118496 Coding 101803 tccagagccaggtagaactg 71 56 118497 Coding 10 1860cagcggctacagaggtagga 53 57 118499 Coding 10 1925 ttccatcttcatcaagcatg 059 118501 Coding 10 1966 catttttcggagtgctgcca 62 61 118502 Coding 102016 tctgacaatttacctgtttg 33 62 118503 Coding 10 2029tgttgtcaaaaactctgaca 32 63 118504 Coding 10 2034 caagatgttgtcaaaaactc 064 118505 Coding 10 2067 ggtccagggtccatgaagct 44 65 118506 Coding 102078 gcttaccctcaggtccaggg 73 66 118507 Coding 10 2098atcataatcttcactgtaca 49 67 118508 Coding 10 2179 ttcatcaccacagcgagcat 7268 118509 Coding 10 2267 accgatctagccctcccttc 64 69 118510 Coding 102310 accaaggacattaagtcgca 45 70 118511 Coding 10 2353atacatgtgaacattctgta 48 71 118512 Coding 10 2525 ttgaggtctccttcaactgt 4272 118513 Coding 10 2586 ttccagtcaggtcctttcat 65 73 118514 Coding 102736 gtgcaggcctcatcaagtgc 0 74 118515 Coding 10 2782tggaggaagtcctactgtca 42 75 118516 Coding 10 2854 actggcttcatctatcagct 6976 118517 Coding 10 2897 ccattatttcctgtgtaagg 41 77 118518 Coding 102986 tgccataacttgtatgatca 52 78 118519 Coding 10 3033tctgtggcttcctcagctga 47 79 118520 Coding 10 3053 gactgagattcatcaggccc 3180 118521 Coding 10 3090 ctgagaatgtgatgcaggag 57 81 118522 Coding 103117 cttcgttccactccaaactc 4 82 118523 Coding 10 3137ttgaatcagtgggacgaacg 18 83 118524 Coding 10 3183 gctccaacagcaccaatctc 3684 118525 Coding 10 3225 tcactttttaactgcatgat 35 85 118526 Coding 103245 gaaattccacctgctttatc 19 86 118527 Coding 10 3406ttgacgactatctttagatg 34 87 118528 Coding 10 3576 gagaatttaatctcacctgg 3388 118529 Coding 10 3600 ttcaggacagactccacatg 58 89 118530 Coding 103651 acaaggatggcttcaaccag 53 90 118531 Coding 10 3672atatctgccagcatggtgag 54 91 118532 Coding 10 3682 atgaatttcaatatctgcca 6092 118533 Coding 10 3750 tgttcttgaaggaacaagtc 35 93 118534 Coding 103780 aacatggtatcatctgcgcc 60 94 118535 Coding 10 3790atcctttgccaacatggtat 19 95 118536 Coding 10 3813 agagtacagatgccagatgc 3696 118537 Coding 10 3838 gccactgggtgcactgtcat 36 97 118538 Coding 103861 aggtaggtcatggtgccaaa 59 98 118539 Coding 10 3894aactcctgcacgtaggtggc 46 99

As shown in Table 2, SEQ ID NOs 26, 27, 28, 29, 30, 31, 32, 33, 34, 37,38, 39, 41, 42, 43, 44, 46, 49, 50, 52, 54, 55, 56, 57, 61, 62, 63, 65,66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 81, 84, 85, 87,88, 89, 90, 91, 92, 93, 94, 96, 97, 98 and 99 demonstrated at least 30%inhibition of mouse Phosphorylase kinase alpha 1 expression in thisexperiment and are therefore preferred. The target sites to which thesepreferred sequences are complementary are herein referred to as “activesites” and are therefore preferred sites for targeting by compounds ofthe present invention.

Example 17 Antisense Inhibition of Rat Phosphorylase Kinase Alpha 1Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

In accordance with the present invention, a third series ofoligonucleotides were designed to target different regions of the ratPhosphorylase kinase alpha 1 RNA, using published sequences (GenBankaccession number M92918, incorporated herein as SEQ ID NO: 17). Theoligonucleotides are shown in Table 3. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 3 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on ratPhosphorylase kinase alpha 1 mRNA levels by quantitative real-time PCRas described in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”.

TABLE 3 Inhibition of rat Phosphorylase kinase alpha 1 mRNA levels bychimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap ISIS TARGET TARGET SEQ ID # REGION SEQ ID NO SITE SEQUENCE %INHIB NO 118498 Coding 17 29 ggtgggctggcctgtcatcc 83 58 118504 Coding 17181 caagatgttgtcaaaaactc 73 64 118545 Coding 17 5 ccggctgcagaggtaggaga88 103 118546 Coding 17 9 tccaccggctgcagaggtag 85 104 118547 Coding 1712 tcctccaccggctgcagagg 90 105 118548 Coding 17 16 gtcatcctccaccggctgca96 106 118549 Coding 17 26 gggctggcctgtcatcctcc 91 107 118550 Coding 1735 agtgatggtgggctggcctg 84 108 118551 Coding 17 37 aaagtgatggtgggctggcc86 109 118552 Coding 17 58 agcatggtgtgtgagatggg 45 11o 118553 Coding 1759 cagcatggtgtgtgagatgg 84 111 118554 Coding 17 63 catccagcatggtgtgtgag90 112 118555 Coding 17 66 cttcatccagcatggtgtgt 94 113 118556 Coding 1767 tcttcatccagcatggtgtg 95 114 118557 Coding 17 71 tccatcttcatccagcatgg93 115 118558 Coding 17 74 ggctccatcttcatccagca 93 116 118559 Coding 1777 gctggctccatcttcatcca 95 117 118560 Coding 17 80 caagctggctccatcttcat96 118 118561 Coding 17 87 ttgaattcaagctggctcca 89 119 118562 Coding 1793 ggatacttgaattcaagctg 88 120 118563 Coding 17 95 caggatacttgaattcaagc93 121 118564 Coding 17 96 ccaggatacttgaattcaag 93 122 118565 Coding 1798 tgccaggatacttgaattca 93 123 118566 Coding 17 99 ctgccaggatacttgaattc84 124 118567 Coding 17 100 gctgccaggatacttgaatt 93 125 118568 Coding 17101 tgctgccaggatacttgaat 91 126 118569 Coding 17 104gagtgctgccaggatacttg 92 127 118570 Coding 17 105 ggagtgctgccaggatactt 93128 118571 Coding 17 108 tccggagtgctgccaggata 89 129 118572 Coding 17111 ttttccggagtgctgccagg 94 130 118573 Coding 17 113cattttccggagtgctgcca 91 131 118574 Coding 17 114 gcattttccggagtgctgcc 94132 118575 Coding 17 117 cctgcattttccggagtgct 98 133 118576 Coding 17120 catcctgcattttccggagt 98 134 118577 Coding 17 123aaccatcctgcattttccgg 99 135 118578 Coding 17 126 aataaccatcctgcattttc 91136 118579 Coding 17 129 caaaataaccatcctgcatt 81 137 118580 Coding 17131 accaaaataaccatcctgca 86 138 118581 Coding 17 133ccaccaaaataaccatcctg 82 139 118582 Coding 17 137 ggccccaccaaaataaccat 94140 118583 Coding 17 142 atcctggccccaccaaaata 84 141 118584 Coding 17147 tttggatcctggccccacca 89 142 118585 Coding 17 151cctgtttggatcctggcccc 93 143 118586 Coding 17 154 ttacctgtttggatcctggc 88144 118587 Coding 17 157 agcttacctgtttggatcct 90 145 118588 Coding 17160 gacagcttacctgtttggat 88 146 118589 Coding 17 163tctgacagcttacctgtttg 87 147 118590 Coding 17 166 aactctgacagcttacctgt 80148 118591 Coding 17 169 aaaaactctgacagcttacc 91 149 118592 Coding 17172 gtcaaaaactctgacagctt 82 150 118593 Coding 17 175gttgtcaaaaactctgacag 87 151 118594 Coding 17 178 gatgttgtcaaaaactctga 87152 118595 Coding 17 184 cagcaagatgttgtcaaaaa 89 153 118596 Coding 17187 gtgcagcaagatgttgtcaa 89 154 118597 Coding 17 190tgtgtgcagcaagatgttgt 75 155 118598 Coding 17 201 tgaagcttaagtgtgtgcag 84156 118599 Coding 17 208 gggtccatgaagcttaagtg 73 157 118600 Coding 17212 tccagggtccatgaagctta 93 158 118601 Coding 17 241taatcttcgctgtacagctt 78 159 118602 Coding 17 251 gtcttcatcataatcttcgc 94160 118603 Coding 17 253 tagtcttcatcataatcttc 88 161 118605 Coding 17256 tcatagtcttcatcataatc 76 162 118606 Coding 17 262tcatcatcatagtcttcatc 90 163 118607 Coding 17 267 ccagctcatcatcatagtct 92164 118608 Coding 17 269 gtccagctcatcatcatagt 96 165 118609 Coding 17270 agtccagctcatcatcatag 95 166 118610 Coding 17 272agagtccagctcatcatcat 89 167 118611 Coding 17 276 tgccagagtccagctcatca 94168 118612 Coding 17 283 atccagttgccagagtccag 92 169 118613 Coding 17284 catccagttgccagagtcca 91 170 118614 Coding 17 287atccatccagttgccagagt 91 171 118615 Coding 17 290 gctatccatccagttgccag 83172 118616 Coding 17 294 catagctatccatccagttg 89 173 118617 Coding 17298 gagtcatagctatccatcca 91 174 118618 Coding 17 304cgtgttgagtcatagctatc 78 175 118619 Coding 17 305 acgtgttgagtcatagctat 67176 118620 Coding 17 309 cattacgtgttgagtcatag 80 177 118621 Coding 17310 gcattacgtgttgagtcata 79 178

As shown in Table 3, SEQ ID NOs 58, 64, 103, 104, 105, 106, 107, 108,109, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 and 178demonstrated at least 60% inhibition of rat Phosphorylase kinase alpha 1expression in this experiment and are therefore preferred. The targetsites to which these preferred sequences are complementary are hereinreferred to as “active sites” and are therefore preferred sites fortargeting by compounds of the present invention.

Example 18 Western Blot Analysis of Phosphorylase Kinase Alpha 1 proteinLevels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to Phosphorylase kinasealpha 1 is used, with a radiolabelled or fluorescently labeled secondaryantibody directed against the primary antibody species. Bands arevisualized using a PHOSPHORIMAGER™ (Molecular Dynamics, SunnyvaleCalif.)

178 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcgctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2atgcattctg cccccaagga 20 3 4215 DNA Homo sapiens CDS (162)...(3833) 3gccgccgggc gccaggcctg agcggtggga gggctctgcg gggcctggtg ttcaggcgtc 60ccaccacgag ggtggagcag cgttggatac ttgttcctta gggaccgaag ctccggtggc 120acccgggcta tttctcagag gacaattagt aacgtgtcgc c atg agg agc cgg agt 176Met Arg Ser Arg Ser 1 5 aac tcc ggg gtc cgg ctg gac ggc tac gct cga ctggtg caa cag acc 224 Asn Ser Gly Val Arg Leu Asp Gly Tyr Ala Arg Leu ValGln Gln Thr 10 15 20 atc ctg tgc cat cag aat cca gtg act ggc ttg ctt ccagcc agc tat 272 Ile Leu Cys His Gln Asn Pro Val Thr Gly Leu Leu Pro AlaSer Tyr 25 30 35 gat cag aaa gat gct tgg gtc cga gat aat gtg tac agc atcttg gct 320 Asp Gln Lys Asp Ala Trp Val Arg Asp Asn Val Tyr Ser Ile LeuAla 40 45 50 gtg tgg ggt ttg ggc ctg gcc tat cgg aag aat gca gac cgg gatgag 368 Val Trp Gly Leu Gly Leu Ala Tyr Arg Lys Asn Ala Asp Arg Asp Glu55 60 65 gat aag gca aag gcc tat gaa ttg gag cag agt gta gtg aag ctg atg416 Asp Lys Ala Lys Ala Tyr Glu Leu Glu Gln Ser Val Val Lys Leu Met 7075 80 85 aga gga cta ctg cac tgc atg atc aga cag gtg gat aaa gta gaa tcc464 Arg Gly Leu Leu His Cys Met Ile Arg Gln Val Asp Lys Val Glu Ser 9095 100 ttc aaa tat agt cag agt act aag gat agc ctc cat gca aag tac aac512 Phe Lys Tyr Ser Gln Ser Thr Lys Asp Ser Leu His Ala Lys Tyr Asn 105110 115 acc aaa acc tgt gcc act gta gtg ggt gat gat caa tgg gga cac ctg560 Thr Lys Thr Cys Ala Thr Val Val Gly Asp Asp Gln Trp Gly His Leu 120125 130 cag ttg gat gct acc tct gtg tac ctg ctc ttc tta gcc caa atg act608 Gln Leu Asp Ala Thr Ser Val Tyr Leu Leu Phe Leu Ala Gln Met Thr 135140 145 gcc tca gga ctc cat atc atc cac agc cta gat gaa gtc aat ttc ata656 Ala Ser Gly Leu His Ile Ile His Ser Leu Asp Glu Val Asn Phe Ile 150155 160 165 cag aac ctt gtg ttt tac att gaa gct gca tat aaa act gct gacttc 704 Gln Asn Leu Val Phe Tyr Ile Glu Ala Ala Tyr Lys Thr Ala Asp Phe170 175 180 ggg ata tgg gaa cgt gga gac aag acc aac caa ggg atc tca gagttg 752 Gly Ile Trp Glu Arg Gly Asp Lys Thr Asn Gln Gly Ile Ser Glu Leu185 190 195 aat gcc agt tca gtt gga atg gca aag gca gcc ctg gaa gca ttagat 800 Asn Ala Ser Ser Val Gly Met Ala Lys Ala Ala Leu Glu Ala Leu Asp200 205 210 gaa ctg gat ctg ttt ggt gtg aaa ggt ggg cct caa tca gtt atccat 848 Glu Leu Asp Leu Phe Gly Val Lys Gly Gly Pro Gln Ser Val Ile His215 220 225 gtc ctg gct gat gaa gta cag cac tgc cag tct atc cta aat tcacta 896 Val Leu Ala Asp Glu Val Gln His Cys Gln Ser Ile Leu Asn Ser Leu230 235 240 245 ctg ccc cgt gct tca aca tca aaa gag gtt gat gct agt ctactc tca 944 Leu Pro Arg Ala Ser Thr Ser Lys Glu Val Asp Ala Ser Leu LeuSer 250 255 260 gtg gtt tcc ttc cct gcc ttt gca gta gag gat agc cag ttggtg gag 992 Val Val Ser Phe Pro Ala Phe Ala Val Glu Asp Ser Gln Leu ValGlu 265 270 275 ctc aca aaa cag gaa atc atc acc aag ctt cag ggt cgt tatggt tgc 1040 Leu Thr Lys Gln Glu Ile Ile Thr Lys Leu Gln Gly Arg Tyr GlyCys 280 285 290 tgt cgc ttt cta cga gat gga tat aaa act cct aaa gag gatccc aat 1088 Cys Arg Phe Leu Arg Asp Gly Tyr Lys Thr Pro Lys Glu Asp ProAsn 295 300 305 cgt ctg tac tat gaa cca gct gag ctg aag cta ttt gaa aacatt gag 1136 Arg Leu Tyr Tyr Glu Pro Ala Glu Leu Lys Leu Phe Glu Asn IleGlu 310 315 320 325 tgt gaa tgg cca ttg ttc tgg aca tac ttt att ctt gatggg gtc ttc 1184 Cys Glu Trp Pro Leu Phe Trp Thr Tyr Phe Ile Leu Asp GlyVal Phe 330 335 340 agt ggc aat gca gaa cag gtt caa gaa tat aaa gag gctctt gaa gca 1232 Ser Gly Asn Ala Glu Gln Val Gln Glu Tyr Lys Glu Ala LeuGlu Ala 345 350 355 gtc ctc atc aag ggc aaa aat gga gtc cca ctt ctg ccagag ctg tac 1280 Val Leu Ile Lys Gly Lys Asn Gly Val Pro Leu Leu Pro GluLeu Tyr 360 365 370 agt gtt cct cct gac agg gtc gat gaa gaa tat cag aatcct cac act 1328 Ser Val Pro Pro Asp Arg Val Asp Glu Glu Tyr Gln Asn ProHis Thr 375 380 385 gtg gac cga gtc ccc atg ggg aaa ttg cct cac atg tggggt cag tct 1376 Val Asp Arg Val Pro Met Gly Lys Leu Pro His Met Trp GlyGln Ser 390 395 400 405 cta tac att tta gga agc ttg atg gca gag gga ttttta gcc cct gga 1424 Leu Tyr Ile Leu Gly Ser Leu Met Ala Glu Gly Phe LeuAla Pro Gly 410 415 420 gaa att gat ccc ctg aat cgc agg ttt tct act gtaccg aag ccc gat 1472 Glu Ile Asp Pro Leu Asn Arg Arg Phe Ser Thr Val ProLys Pro Asp 425 430 435 gtt gtg gtt caa gtc tcc att cta gct gaa aca gaagaa atc aag acc 1520 Val Val Val Gln Val Ser Ile Leu Ala Glu Thr Glu GluIle Lys Thr 440 445 450 att ttg aag gac aag gga att tac gtg gag acc attgct gag gta tac 1568 Ile Leu Lys Asp Lys Gly Ile Tyr Val Glu Thr Ile AlaGlu Val Tyr 455 460 465 ccc atc aga gta caa cca gct cgt att ctc agc cacatt tat tcc agc 1616 Pro Ile Arg Val Gln Pro Ala Arg Ile Leu Ser His IleTyr Ser Ser 470 475 480 485 cta gga tgc aac aat aga atg aaa ctc agt ggacga ccc tac aga cac 1664 Leu Gly Cys Asn Asn Arg Met Lys Leu Ser Gly ArgPro Tyr Arg His 490 495 500 atg gga gtg ctt gga act tca aaa ctc tat gacatt cgg aaa act atc 1712 Met Gly Val Leu Gly Thr Ser Lys Leu Tyr Asp IleArg Lys Thr Ile 505 510 515 ttt act ttc act cca cag ttt ata gac cag caacag ttc tac ctg gct 1760 Phe Thr Phe Thr Pro Gln Phe Ile Asp Gln Gln GlnPhe Tyr Leu Ala 520 525 530 ctg gac aac aag atg ata gtg gaa atg ctt agaaca gac ctc tcc tac 1808 Leu Asp Asn Lys Met Ile Val Glu Met Leu Arg ThrAsp Leu Ser Tyr 535 540 545 ctc tgt agc cgc tgg cgg atg aca ggc cag cccacc atc acc ttc ccc 1856 Leu Cys Ser Arg Trp Arg Met Thr Gly Gln Pro ThrIle Thr Phe Pro 550 555 560 565 atc tca tac agc atg ctt gat gaa gat ggaaca agc ttg aat tca agt 1904 Ile Ser Tyr Ser Met Leu Asp Glu Asp Gly ThrSer Leu Asn Ser Ser 570 575 580 atc ctg gca gca ctc cga aaa atg caa gatggg tat ttt ggt ggg gca 1952 Ile Leu Ala Ala Leu Arg Lys Met Gln Asp GlyTyr Phe Gly Gly Ala 585 590 595 agg gtt caa aca ggt aaa ttg tca gag tttttg aca aca tct tgt tgc 2000 Arg Val Gln Thr Gly Lys Leu Ser Glu Phe LeuThr Thr Ser Cys Cys 600 605 610 aca cac ttg agc ttc atg gac cct gga cctgag ggt aag ctg tac agt 2048 Thr His Leu Ser Phe Met Asp Pro Gly Pro GluGly Lys Leu Tyr Ser 615 620 625 gaa gat tat gat gac aac tat gat tac ctggaa tct ggc aac tgg atg 2096 Glu Asp Tyr Asp Asp Asn Tyr Asp Tyr Leu GluSer Gly Asn Trp Met 630 635 640 645 aat gat tat gat tca acc agt cat gctcgc tgt ggt gat gaa gtt gct 2144 Asn Asp Tyr Asp Ser Thr Ser His Ala ArgCys Gly Asp Glu Val Ala 650 655 660 cgt tat tta gat cac ctt ttg gcg cacact gct ccc cat cct aaa cta 2192 Arg Tyr Leu Asp His Leu Leu Ala His ThrAla Pro His Pro Lys Leu 665 670 675 gcc cct acc tca cag aag gga ggg ctagat cgg ttc caa gct gct gtg 2240 Ala Pro Thr Ser Gln Lys Gly Gly Leu AspArg Phe Gln Ala Ala Val 680 685 690 caa aca acc tgc gac tta atg tcc ttggtg acc aag gcc aag gaa ctg 2288 Gln Thr Thr Cys Asp Leu Met Ser Leu ValThr Lys Ala Lys Glu Leu 695 700 705 cat gta cag aat gtt cac atg tat cttcct acg aag tta ttt cag gct 2336 His Val Gln Asn Val His Met Tyr Leu ProThr Lys Leu Phe Gln Ala 710 715 720 725 tcc cgg cct tca ttc aac tta cttgat tca cct cat ccc cga cag gag 2384 Ser Arg Pro Ser Phe Asn Leu Leu AspSer Pro His Pro Arg Gln Glu 730 735 740 aac cag gtt ccc tct gtt cgt gtagaa ata cat ctt cct aga gac cag 2432 Asn Gln Val Pro Ser Val Arg Val GluIle His Leu Pro Arg Asp Gln 745 750 755 tct ggg gag gtg gac ttt aaa gcactg gtt tta cag ttg aag gag acc 2480 Ser Gly Glu Val Asp Phe Lys Ala LeuVal Leu Gln Leu Lys Glu Thr 760 765 770 tca agc tta cag gaa caa gct gatatc ctc tat atg ctg tat act atg 2528 Ser Ser Leu Gln Glu Gln Ala Asp IleLeu Tyr Met Leu Tyr Thr Met 775 780 785 aaa gga cct gac tgg aac act gaattg tat aat gaa cgg agt gct aca 2576 Lys Gly Pro Asp Trp Asn Thr Glu LeuTyr Asn Glu Arg Ser Ala Thr 790 795 800 805 gtg aga gag ctt ctt acc gagctg tat ggc aaa gtg gga gaa att cgt 2624 Val Arg Glu Leu Leu Thr Glu LeuTyr Gly Lys Val Gly Glu Ile Arg 810 815 820 cac tgg ggc ctg atc cga tacatt tct ggg atc tta agg aag aaa gtg 2672 His Trp Gly Leu Ile Arg Tyr IleSer Gly Ile Leu Arg Lys Lys Val 825 830 835 gaa gca ctt gat gag gcc tgcaca gac ctt ctc tcc cac cag aaa cat 2720 Glu Ala Leu Asp Glu Ala Cys ThrAsp Leu Leu Ser His Gln Lys His 840 845 850 ttg aca gta gga ctt cct ccagaa cct cga gaa aag act atc tct gca 2768 Leu Thr Val Gly Leu Pro Pro GluPro Arg Glu Lys Thr Ile Ser Ala 855 860 865 cct ctg ccc tat gag gcg ctcact cag ctg ata gat gaa gcc agt gaa 2816 Pro Leu Pro Tyr Glu Ala Leu ThrGln Leu Ile Asp Glu Ala Ser Glu 870 875 880 885 ggg gat atg agc att tcaatc ctt aca cag gaa ata atg gta tat cta 2864 Gly Asp Met Ser Ile Ser IleLeu Thr Gln Glu Ile Met Val Tyr Leu 890 895 900 gcc atg tat atg cga acccag cct ggc ctc ttt gct gaa atg ttt cga 2912 Ala Met Tyr Met Arg Thr GlnPro Gly Leu Phe Ala Glu Met Phe Arg 905 910 915 ctt cga att ggt ctg atcata caa gtt atg gca aca gaa ctg gcc cac 2960 Leu Arg Ile Gly Leu Ile IleGln Val Met Ala Thr Glu Leu Ala His 920 925 930 tcc ctt cga tgc tca gctgag gaa gcc aca gag ggc ctg atg aat ctc 3008 Ser Leu Arg Cys Ser Ala GluGlu Ala Thr Glu Gly Leu Met Asn Leu 935 940 945 agt cct tcg gcc atg aagaat ctc ctg cat cac att ctc agc ggc aag 3056 Ser Pro Ser Ala Met Lys AsnLeu Leu His His Ile Leu Ser Gly Lys 950 955 960 965 gag ttt gga gtg gaacga agc gtt cgt ccc act gat tca aat gtc agt 3104 Glu Phe Gly Val Glu ArgSer Val Arg Pro Thr Asp Ser Asn Val Ser 970 975 980 cct gct att tct atccac gag att ggt gct gtt gga gca acc aaa aca 3152 Pro Ala Ile Ser Ile HisGlu Ile Gly Ala Val Gly Ala Thr Lys Thr 985 990 995 gaa cga act ggg atcatg cag tta aaa agt gag ata aag cag gtg gaa 3200 Glu Arg Thr Gly Ile MetGln Leu Lys Ser Glu Ile Lys Gln Val Glu 1000 1005 1010 ttt cgt aga ctgtca atc tca gct gag agt cag tca cct gga acc tct 3248 Phe Arg Arg Leu SerIle Ser Ala Glu Ser Gln Ser Pro Gly Thr Ser 1015 1020 1025 atg act ccaagt agt ggg tcc ttt cct agt gca tat gat cag cag tca 3296 Met Thr Pro SerSer Gly Ser Phe Pro Ser Ala Tyr Asp Gln Gln Ser 1030 1035 1040 1045 tctaaa gat agt cgt caa ggt caa tgg caa cgc cga aga agg ctg gat 3344 Ser LysAsp Ser Arg Gln Gly Gln Trp Gln Arg Arg Arg Arg Leu Asp 1050 1055 1060ggg gca ctg aat aga gtt cca gtt gga ttt tat cag aaa gta tgg aaa 3392 GlyAla Leu Asn Arg Val Pro Val Gly Phe Tyr Gln Lys Val Trp Lys 1065 10701075 gtt ttg cag aag tgt cac gga ctt tct gtt gaa ggg ttt gtc ctt cct3440 Val Leu Gln Lys Cys His Gly Leu Ser Val Glu Gly Phe Val Leu Pro1080 1085 1090 tcc tct acc act aga gag atg act cca ggt gag att aaa ttctct gtt 3488 Ser Ser Thr Thr Arg Glu Met Thr Pro Gly Glu Ile Lys Phe SerVal 1095 1100 1105 cat gtg gag tct gtc ctg aat cgt gta cct cag cca gagtac cgt cag 3536 His Val Glu Ser Val Leu Asn Arg Val Pro Gln Pro Glu TyrArg Gln 1110 1115 1120 1125 ctg ctg gtt gaa gcc atc ctt gtc ctc acc atgctg gca gat att gaa 3584 Leu Leu Val Glu Ala Ile Leu Val Leu Thr Met LeuAla Asp Ile Glu 1130 1135 1140 att cat agc atc gga agc atc att gct gtggaa aaa ata gtg cat att 3632 Ile His Ser Ile Gly Ser Ile Ile Ala Val GluLys Ile Val His Ile 1145 1150 1155 gcc aat gac ttg ttc ctt caa gaa cagaaa acc ctt ggc gca gat gat 3680 Ala Asn Asp Leu Phe Leu Gln Glu Gln LysThr Leu Gly Ala Asp Asp 1160 1165 1170 acc atg ttg gca aag gat ccc gcatct ggc atc tgt act ctt ctg tat 3728 Thr Met Leu Ala Lys Asp Pro Ala SerGly Ile Cys Thr Leu Leu Tyr 1175 1180 1185 gac agt gca ccc agt ggc aggttt ggc acc atg acc tac ctc tcc aag 3776 Asp Ser Ala Pro Ser Gly Arg PheGly Thr Met Thr Tyr Leu Ser Lys 1190 1195 1200 1205 gca gcc gcc acc tacgtg cag gag ttc ctg ccc cac agc atc tgt gcc 3824 Ala Ala Ala Thr Tyr ValGln Glu Phe Leu Pro His Ser Ile Cys Ala 1210 1215 1220 atg caa tgagggctttggt tcctggcttc tgggagcctt ttgacagctg gtccctgcct 3883 Met Glncggttgattg tgcatggaac taaaatgtta ttgcctaatc actccaaccc tgcccctttc 3943tgtcccatcc ttcccaagaa gagagaactt tttcgataaa ctaactactg tagaagaagt 4003gaacacttac ctggaggctc accttgcaga accagtgaca atcttatgag tataatgaac 4063actcagccag gcctgtcatg attggcttta tttctttcat cattcataaa agtttgcatg 4123tgtttttatt ctctagatct gttaccaata tagttttcta actcctgttt ggggagcaag 4183tgttaataat aacttattcc taaaaaaaaa aa 4215 4 21 DNA Artificial SequencePCR Primer 4 gacttcggga tatgggaacg t 21 5 23 DNA Artificial Sequence PCRPrimer 5 tcacaccaaa cagatccagt tca 23 6 28 DNA Artificial Sequence PCRProbe 6 aaccaaggga tctcagagtt gaatgcca 28 7 21 DNA Artificial SequencePCR Primer 7 caacggattt ggtcgtattg g 21 8 26 DNA Artificial Sequence PCRPrimer 8 ggcaacaata tccactttac cagagt 26 9 21 DNA Artificial SequencePCR Probe 9 cgcctggtca ccagggctgc t 21 10 4130 DNA Mus musculus CDS(219)...(3944) 10 cggggaccac gggagactga gtggagacgg ggtctttgcc ggccgaagcctagaccgcgg 60 gaggctgagc cgcggaatga agtggactct gaggccgccg gaaccaggtgacgcgcggga 120 accattggcc acacgagcaa gggagcgttt agaaggtttc cgtaggcctggcgtccgagc 180 ttcccgtgtg tgcagtgggg gattagggac gtggcacc atg agg agc cgcagt aac 236 Met Arg Ser Arg Ser Asn 1 5 tcg gga gtc cgc ctg gac ggc tatgct cgg ctg gtg cat cag acc atc 284 Ser Gly Val Arg Leu Asp Gly Tyr AlaArg Leu Val His Gln Thr Ile 10 15 20 ctg tgc cat cag aat cca gtg aca ggcttg ctt cca gcc agc tat gat 332 Leu Cys His Gln Asn Pro Val Thr Gly LeuLeu Pro Ala Ser Tyr Asp 25 30 35 caa aaa gat gcc tgg gtc cga gat aat gtgtac agt atc ctg gct gtg 380 Gln Lys Asp Ala Trp Val Arg Asp Asn Val TyrSer Ile Leu Ala Val 40 45 50 tgg ggt ttg ggc ctg gca tat cgc aaa aat gctgac cgt gat gag gac 428 Trp Gly Leu Gly Leu Ala Tyr Arg Lys Asn Ala AspArg Asp Glu Asp 55 60 65 70 aag gca aag gcc tat gaa ctg gag cag agt gtagtg aag tta atg agg 476 Lys Ala Lys Ala Tyr Glu Leu Glu Gln Ser Val ValLys Leu Met Arg 75 80 85 gga ctg ctg cac tgc atg atc aga cag gtg gat aaagta gag tcc ttc 524 Gly Leu Leu His Cys Met Ile Arg Gln Val Asp Lys ValGlu Ser Phe 90 95 100 aag tac agt cag agt act ccc gat agc ctc cat gccaag tac aac acc 572 Lys Tyr Ser Gln Ser Thr Pro Asp Ser Leu His Ala LysTyr Asn Thr 105 110 115 aaa act tgt gcc act gtg gtg ggt gat gac cag tgggga cac ctg cag 620 Lys Thr Cys Ala Thr Val Val Gly Asp Asp Gln Trp GlyHis Leu Gln 120 125 130 ttg gat gct act tct gtg tac ctg ctc ttc cta gcacaa atg act gcc 668 Leu Asp Ala Thr Ser Val Tyr Leu Leu Phe Leu Ala GlnMet Thr Ala 135 140 145 150 tca gga ctc cat atc atc cac agc cta gac gaagtc aat ttt ata cag 716 Ser Gly Leu His Ile Ile His Ser Leu Asp Glu ValAsn Phe Ile Gln 155 160 165 aac ctc gtg ttt tac att gaa gct gca tat aaaact gct gac ttt ggg 764 Asn Leu Val Phe Tyr Ile Glu Ala Ala Tyr Lys ThrAla Asp Phe Gly 170 175 180 ata tgg gaa cgt ggc gat aag aca aac caa ggcatc tcg gaa ttg aat 812 Ile Trp Glu Arg Gly Asp Lys Thr Asn Gln Gly IleSer Glu Leu Asn 185 190 195 gcg agt tca gtt gga atg gcc aag gca gcc ctggaa gca cta gat gaa 860 Ala Ser Ser Val Gly Met Ala Lys Ala Ala Leu GluAla Leu Asp Glu 200 205 210 tta gac ttg ttt ggt gtg aaa ggt ggg cca caatca gtt atc cat gtc 908 Leu Asp Leu Phe Gly Val Lys Gly Gly Pro Gln SerVal Ile His Val 215 220 225 230 ctg gct gat gaa gtc caa cac tgc cag tctatc ctg aat tca cta ctg 956 Leu Ala Asp Glu Val Gln His Cys Gln Ser IleLeu Asn Ser Leu Leu 235 240 245 ccc agg gct tca aca tcc aaa gaa gtt gatgcc agt ctg ctc tca gtg 1004 Pro Arg Ala Ser Thr Ser Lys Glu Val Asp AlaSer Leu Leu Ser Val 250 255 260 gtc tct ttc cca gcc ttt gct gta gag gacagc cat ttg gtg gag ctc 1052 Val Ser Phe Pro Ala Phe Ala Val Glu Asp SerHis Leu Val Glu Leu 265 270 275 acc aaa cag gag atc atc acc aag ctt cagggt cgt tat ggt tgc tgt 1100 Thr Lys Gln Glu Ile Ile Thr Lys Leu Gln GlyArg Tyr Gly Cys Cys 280 285 290 cgt ttt ctg cga gat gga tat aaa act cctaaa gag gat ccc caa cgc 1148 Arg Phe Leu Arg Asp Gly Tyr Lys Thr Pro LysGlu Asp Pro Gln Arg 295 300 305 310 cta tac tat aac cca gct gag ctg aagcta ttt gaa aac att gag tgc 1196 Leu Tyr Tyr Asn Pro Ala Glu Leu Lys LeuPhe Glu Asn Ile Glu Cys 315 320 325 gaa tgg cca ttg ttc tgg aca tac tttatc ctt gat ggg atc ttc agt 1244 Glu Trp Pro Leu Phe Trp Thr Tyr Phe IleLeu Asp Gly Ile Phe Ser 330 335 340 ggc aac gta gaa cag gtt caa gaa tataga gag gct ctt gat gca gtc 1292 Gly Asn Val Glu Gln Val Gln Glu Tyr ArgGlu Ala Leu Asp Ala Val 345 350 355 ctc atc aag ggc aaa aat gga gtc cctctt ctt cca gag ctg tac agt 1340 Leu Ile Lys Gly Lys Asn Gly Val Pro LeuLeu Pro Glu Leu Tyr Ser 360 365 370 gtc cct cct gac agg gtt gat gaa gagtat caa aat ccc cac act gtg 1388 Val Pro Pro Asp Arg Val Asp Glu Glu TyrGln Asn Pro His Thr Val 375 380 385 390 gat cga gtc cct atg gga aaa ttgcct cac atg tgg ggt cag tct cta 1436 Asp Arg Val Pro Met Gly Lys Leu ProHis Met Trp Gly Gln Ser Leu 395 400 405 tac att tta gga agc ttg atg gcagag gga ttt tta gct cct gga gaa 1484 Tyr Ile Leu Gly Ser Leu Met Ala GluGly Phe Leu Ala Pro Gly Glu 410 415 420 att gat ccc ctg aat cgt agg ttttct act gtg cca aag cca gat gtg 1532 Ile Asp Pro Leu Asn Arg Arg Phe SerThr Val Pro Lys Pro Asp Val 425 430 435 gtg gtt caa gtc tcc att ctg gctgaa aca gaa gaa atc aag gcc att 1580 Val Val Gln Val Ser Ile Leu Ala GluThr Glu Glu Ile Lys Ala Ile 440 445 450 ttg aag gac aaa gga att gat gtggag acc att gct gaa gtg tac ccc 1628 Leu Lys Asp Lys Gly Ile Asp Val GluThr Ile Ala Glu Val Tyr Pro 455 460 465 470 ata aga gta cag cca gct cgtatt ctc agc cat att tat tct agt cta 1676 Ile Arg Val Gln Pro Ala Arg IleLeu Ser His Ile Tyr Ser Ser Leu 475 480 485 gga tgc aac agt aga atg aaactc agt gga cga ccc tac agg ctc atg 1724 Gly Cys Asn Ser Arg Met Lys LeuSer Gly Arg Pro Tyr Arg Leu Met 490 495 500 ggt gtg ctt gga aca tca aaactt tat gac att cgc aaa act atc ttt 1772 Gly Val Leu Gly Thr Ser Lys LeuTyr Asp Ile Arg Lys Thr Ile Phe 505 510 515 act ttc act cca cag ttt atagac cag caa cag ttc tac ctg gct ctg 1820 Thr Phe Thr Pro Gln Phe Ile AspGln Gln Gln Phe Tyr Leu Ala Leu 520 525 530 gac aac cag atg ata gta gaaatg ctc aga aca gac ctt tcc tac ctc 1868 Asp Asn Gln Met Ile Val Glu MetLeu Arg Thr Asp Leu Ser Tyr Leu 535 540 545 550 tgt agc cgc tgg agg atgaca ggc cag ccc acg atc act ttc cct atc 1916 Cys Ser Arg Trp Arg Met ThrGly Gln Pro Thr Ile Thr Phe Pro Ile 555 560 565 tcg cac acc atg ctt gatgaa gat gga acc agc ttg aat tca agt atc 1964 Ser His Thr Met Leu Asp GluAsp Gly Thr Ser Leu Asn Ser Ser Ile 570 575 580 ttg gca gca ctc cga aaaatg cag gat ggc tat ttt ggt ggg gcc agg 2012 Leu Ala Ala Leu Arg Lys MetGln Asp Gly Tyr Phe Gly Gly Ala Arg 585 590 595 atc caa aca ggt aaa ttgtca gag ttt ttg aca aca tct tgc tgc aca 2060 Ile Gln Thr Gly Lys Leu SerGlu Phe Leu Thr Thr Ser Cys Cys Thr 600 605 610 cac tta agc ttc atg gaccct gga cct gag ggt aag ctg tac agt gaa 2108 His Leu Ser Phe Met Asp ProGly Pro Glu Gly Lys Leu Tyr Ser Glu 615 620 625 630 gat tat gat gaa gactat gaa gat gat ttg gac tct ggc aac tgg atg 2156 Asp Tyr Asp Glu Asp TyrGlu Asp Asp Leu Asp Ser Gly Asn Trp Met 635 640 645 gac agc tat gat tcaaca agt aat gct cgc tgt ggt gat gaa gtt gcc 2204 Asp Ser Tyr Asp Ser ThrSer Asn Ala Arg Cys Gly Asp Glu Val Ala 650 655 660 cgt tat tta gac cgcctt ttg gca cac act gtt ccc cat cct aaa cta 2252 Arg Tyr Leu Asp Arg LeuLeu Ala His Thr Val Pro His Pro Lys Leu 665 670 675 gct cct acc tca cggaag gga ggg cta gat cgg ttc cga gct gct gtg 2300 Ala Pro Thr Ser Arg LysGly Gly Leu Asp Arg Phe Arg Ala Ala Val 680 685 690 caa aca act tgc gactta atg tcc ttg gtg gcc aag gcc aag gag ctg 2348 Gln Thr Thr Cys Asp LeuMet Ser Leu Val Ala Lys Ala Lys Glu Leu 695 700 705 710 cat ata cag aatgtt cac atg tat att cct aca aag tta ttt cag cct 2396 His Ile Gln Asn ValHis Met Tyr Ile Pro Thr Lys Leu Phe Gln Pro 715 720 725 tct cgt cct tcactc aac tta ctt gac tcc cct gag tct cca caa gat 2444 Ser Arg Pro Ser LeuAsn Leu Leu Asp Ser Pro Glu Ser Pro Gln Asp 730 735 740 agc cag gtt ccttcc gtt cat gta gaa gtg cat ctt cct agg gac cag 2492 Ser Gln Val Pro SerVal His Val Glu Val His Leu Pro Arg Asp Gln 745 750 755 tct ggg gaa gtggac ttc cag tca ttg gtt tca cag ttg aag gag acc 2540 Ser Gly Glu Val AspPhe Gln Ser Leu Val Ser Gln Leu Lys Glu Thr 760 765 770 tca agc tta caggag caa gct gat ata ctc tac atg ctg tat tca atg 2588 Ser Ser Leu Gln GluGln Ala Asp Ile Leu Tyr Met Leu Tyr Ser Met 775 780 785 790 aaa gga cctgac tgg aac act gaa ttg tat gaa gaa ggg ggg gct act 2636 Lys Gly Pro AspTrp Asn Thr Glu Leu Tyr Glu Glu Gly Gly Ala Thr 795 800 805 gtc aga gagctt ctt agt gaa ctg tat gtc aaa gtt ggt gaa att cgg 2684 Val Arg Glu LeuLeu Ser Glu Leu Tyr Val Lys Val Gly Glu Ile Arg 810 815 820 cac tgg ggtctg atc cga tat atc tct ggg atc tta cgg aag aaa gtg 2732 His Trp Gly LeuIle Arg Tyr Ile Ser Gly Ile Leu Arg Lys Lys Val 825 830 835 gag gca cttgat gag gcc tgc aca gac ctt ctg tcc tac cag aaa cac 2780 Glu Ala Leu AspGlu Ala Cys Thr Asp Leu Leu Ser Tyr Gln Lys His 840 845 850 ctg aca gtagga ctt cct cca gaa cct cga gag aag acc atc tct gcg 2828 Leu Thr Val GlyLeu Pro Pro Glu Pro Arg Glu Lys Thr Ile Ser Ala 855 860 865 870 cct ctaccg tac gag gca ctc act aag ctg ata gat gaa gcc agt gaa 2876 Pro Leu ProTyr Glu Ala Leu Thr Lys Leu Ile Asp Glu Ala Ser Glu 875 880 885 ggc gacatg agc atc tca acc ctt aca cag gaa ata atg gtc tat ctt 2924 Gly Asp MetSer Ile Ser Thr Leu Thr Gln Glu Ile Met Val Tyr Leu 890 895 900 gcc atgtat atg aga act cag cct ggc ctc ttt gca gaa atg ttc aga 2972 Ala Met TyrMet Arg Thr Gln Pro Gly Leu Phe Ala Glu Met Phe Arg 905 910 915 ctt cgaatc ggt ttg atc ata caa gtt atg gca aca gaa cta gca cac 3020 Leu Arg IleGly Leu Ile Ile Gln Val Met Ala Thr Glu Leu Ala His 920 925 930 tct cttcga tgt tca gct gag gaa gcc aca gag ggc ctg atg aat ctc 3068 Ser Leu ArgCys Ser Ala Glu Glu Ala Thr Glu Gly Leu Met Asn Leu 935 940 945 950 agtcct tca gcc atg aag aac ctc ctg cat cac att ctc agt ggc aaa 3116 Ser ProSer Ala Met Lys Asn Leu Leu His His Ile Leu Ser Gly Lys 955 960 965 gagttt gga gtg gaa cga agc gtt cgt ccc act gat tca aat gtg agt 3164 Glu PheGly Val Glu Arg Ser Val Arg Pro Thr Asp Ser Asn Val Ser 970 975 980 cctgct att tcc atc cat gag att ggt gct gtt gga gca aca aaa act 3212 Pro AlaIle Ser Ile His Glu Ile Gly Ala Val Gly Ala Thr Lys Thr 985 990 995 gaacga act gga atc atg cag tta aaa agt gag ata aag cag gtg gaa 3260 Glu ArgThr Gly Ile Met Gln Leu Lys Ser Glu Ile Lys Gln Val Glu 1000 1005 1010ttt cgt agg ctg tct gtc tcg atg gag agt cag act agt ggt ggt cat 3308 PheArg Arg Leu Ser Val Ser Met Glu Ser Gln Thr Ser Gly Gly His 1015 10201025 1030 ccc tcg ggt gta gat ttg atg tcg cca tcc ttt ctg tcc cct gcagcc 3356 Pro Ser Gly Val Asp Leu Met Ser Pro Ser Phe Leu Ser Pro Ala Ala1035 1040 1045 tgt att gct gca agt agt gga tcc ttt cct acg gtg tgt gatcat cag 3404 Cys Ile Ala Ala Ser Ser Gly Ser Phe Pro Thr Val Cys Asp HisGln 1050 1055 1060 aca tct aaa gat agt cgt caa ggc cag tgg caa cgc aggaga agg cta 3452 Thr Ser Lys Asp Ser Arg Gln Gly Gln Trp Gln Arg Arg ArgArg Leu 1065 1070 1075 gat gga gca cta aat aga gta cca att gga ttt tatcaa aaa gta tgg 3500 Asp Gly Ala Leu Asn Arg Val Pro Ile Gly Phe Tyr GlnLys Val Trp 1080 1085 1090 aaa att ttg cag aaa tgt cat ggg ctt tct gtggaa ggt ttt gtt ctt 3548 Lys Ile Leu Gln Lys Cys His Gly Leu Ser Val GluGly Phe Val Leu 1095 1100 1105 1110 ccc tct tca acc act agg gag atg acccca ggt gag att aaa ttc tct 3596 Pro Ser Ser Thr Thr Arg Glu Met Thr ProGly Glu Ile Lys Phe Ser 1115 1120 1125 gtc cat gtg gag tct gtc ctg aatcgt gtc cct cag cca gaa tac cgc 3644 Val His Val Glu Ser Val Leu Asn ArgVal Pro Gln Pro Glu Tyr Arg 1130 1135 1140 caa ctt ctg gtt gaa gcc atcctt gtt ctc acc atg ctg gca gat att 3692 Gln Leu Leu Val Glu Ala Ile LeuVal Leu Thr Met Leu Ala Asp Ile 1145 1150 1155 gaa att cat agc att gggagc atc att gct gtg gag aaa ata gtt cat 3740 Glu Ile His Ser Ile Gly SerIle Ile Ala Val Glu Lys Ile Val His 1160 1165 1170 att gcc aac gac ttgttc ctt caa gaa cag aaa acc ctc ggc gca gat 3788 Ile Ala Asn Asp Leu PheLeu Gln Glu Gln Lys Thr Leu Gly Ala Asp 1175 1180 1185 1190 gat acc atgttg gca aag gat cct gca tct ggc atc tgt act ctc ctg 3836 Asp Thr Met LeuAla Lys Asp Pro Ala Ser Gly Ile Cys Thr Leu Leu 1195 1200 1205 tat gacagt gca ccc agt ggc aga ttt ggc acc atg acc tac ctc tcc 3884 Tyr Asp SerAla Pro Ser Gly Arg Phe Gly Thr Met Thr Tyr Leu Ser 1210 1215 1220 aaggca gct gcc acc tac gtg cag gag ttt ctg cca cac agc ctc tgt 3932 Lys AlaAla Ala Thr Tyr Val Gln Glu Phe Leu Pro His Ser Leu Cys 1225 1230 1235gcc atg cag tga gggctgtggg ttctgcctgt tagaacactt cagtagctag 3984 Ala MetGln 1240 cccgtctggt tgattgtgca tggaactgac atgttaacac ctgatcactcccccaggccc 4044 ttttcctgcc ccatccctcc aggaagagag aactttgctg acgatctcactacttttgaa 4104 gaaaagaaga aatatctgga gacttg 4130 11 20 DNA ArtificialSequence PCR Primer 11 gggaacgtgg cgataagaca 20 12 29 DNA ArtificialSequence PCR Primer 12 accaaacaag tctaattcat ctagtgctt 29 13 25 DNAArtificial Sequence PCR Probe 13 ccaaggcatc tcggaattga atgcg 25 14 20DNA Artificial Sequence PCR Primer 14 ggcaaattca acggcacagt 20 15 20 DNAArtificial Sequence PCR Primer 15 gggtctcgct cctggaagct 20 16 27 DNAArtificial Sequence PCR Probe 16 aaggccgaga atgggaagct tgtcatc 27 17 336DNA Rattus norvegicus CDS (1)...(336) 17 gat ctc tcc tac ctc tgc agc cggtgg agg atg aca ggc cag ccc acc 48 Asp Leu Ser Tyr Leu Cys Ser Arg TrpArg Met Thr Gly Gln Pro Thr 1 5 10 15 atc act ttc ccc atc tca cac accatg ctg gat gaa gat gga gcc agc 96 Ile Thr Phe Pro Ile Ser His Thr MetLeu Asp Glu Asp Gly Ala Ser 20 25 30 ttg aat tca agt atc ctg gca gca ctccgg aaa atg cag gat ggt tat 144 Leu Asn Ser Ser Ile Leu Ala Ala Leu ArgLys Met Gln Asp Gly Tyr 35 40 45 ttt ggt ggg gcc agg atc caa aca ggt aagctg tca gag ttt ttg aca 192 Phe Gly Gly Ala Arg Ile Gln Thr Gly Lys LeuSer Glu Phe Leu Thr 50 55 60 aca tct tgc tgc aca cac tta agc ttc atg gaccct gga ccc gag ggt 240 Thr Ser Cys Cys Thr His Leu Ser Phe Met Asp ProGly Pro Glu Gly 65 70 75 80 aag ctg tac agc gaa gat tat gat gaa gac tatgat gat gag ctg gac 288 Lys Leu Tyr Ser Glu Asp Tyr Asp Glu Asp Tyr AspAsp Glu Leu Asp 85 90 95 tct ggc aac tgg atg gat agc tat gac tca aca cgtaat gct cgc tgt 336 Ser Gly Asn Trp Met Asp Ser Tyr Asp Ser Thr Arg AsnAla Arg Cys 100 105 110 18 20 DNA Artificial Sequence PCR Primer 18tgaagatgga gccagcttga 20 19 23 DNA Artificial Sequence PCR Primer 19caccaaaata accatcctgc att 23 20 25 DNA Artificial Sequence PCR Probe 20tcaagtatcc tggcagcact ccgga 25 21 23 DNA Artificial Sequence PCR Primer21 tgttctagag acagccgcat ctt 23 22 21 DNA Artificial Sequence PCR Primer22 caccgacctt caccatcttg t 21 23 24 DNA Artificial Sequence PCR Probe 23ttgtgcagtg ccagcctcgt ctca 24 24 417 DNA Homo sapiens CDS (1)...(60) 24tcc ggg gtc cgg ctg gac ggc tac gct cga ctg gtg caa cag acc atc 48 SerGly Val Arg Leu Asp Gly Tyr Ala Arg Leu Val Gln Gln Thr Ile 1 5 10 15ctg tgc cat cag gtaactgcag gggtgtctct cgtaatcacc gagcgaggat 100 Leu CysHis Gln 20 gtagcttgtc atagcttgga cggagacctt ggatgaactc cgtgcgtgcgtgcgtgtgtg 160 ggtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgac ggagaccttgcatgaactcc 220 gcgtgtgtgt ttgtgtgtgt atctgtatct gtctgtagca ggttctcccgcccctgttaa 280 ttatatattt tttctgctgg tttttcattg ctagttcgct agttcattctaactagaaag 340 gtaaaacttt cttttaataa tacccattat aaatgcactt aataatacctttaattgtgc 400 ctttaataat acccaca 417 25 20 DNA Artificial SequenceAntisense Oligonucleotide 25 cggctcctca tggcgacacg 20 26 20 DNAArtificial Sequence Antisense Oligonucleotide 26 tgatggcaca ggatggtctg20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 tagctggctggaagcaagcc 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28ctgtacacat tatctcggac 20 29 20 DNA Artificial Sequence AntisenseOligonucleotide 29 gcccaaaccc cacacagcca 20 30 20 DNA ArtificialSequence Antisense Oligonucleotide 30 cttcactaca ctctgctcca 20 31 20 DNAArtificial Sequence Antisense Oligonucleotide 31 tgtctgatca tgcagtgcag20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 actttatccacctgtctgat 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33tccaactgca ggtgtcccca 20 34 20 DNA Artificial Sequence AntisenseOligonucleotide 34 agtcctgagg cagtcatttg 20 35 20 DNA ArtificialSequence Antisense Oligonucleotide 35 ctaggctgtg gatgatatgg 20 36 20 DNAArtificial Sequence Antisense Oligonucleotide 36 gttttatatg cagcttcaat20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 cccacctttcacaccaaaca 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38aggacatgga taactgattg 20 39 20 DNA Artificial Sequence AntisenseOligonucleotide 39 ccctgaagct tggtgatgat 20 40 20 DNA ArtificialSequence Antisense Oligonucleotide 40 aaccataacg accctgaagc 20 41 20 DNAArtificial Sequence Antisense Oligonucleotide 41 ttaggagttt tatatccatc20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 aatagcttcagctcagctgg 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43gtccagaaca atggccattc 20 44 20 DNA Artificial Sequence AntisenseOligonucleotide 44 tatattcttg aacctgttct 20 45 20 DNA ArtificialSequence Antisense Oligonucleotide 45 ttgcccttga tgaggactgc 20 46 20 DNAArtificial Sequence Antisense Oligonucleotide 46 ccccacatgt gaggcaattt20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 tagagactgaccccacatgt 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48tccctctgcc atcaagcttc 20 49 20 DNA Artificial Sequence AntisenseOligonucleotide 49 agaatggaga cttgaaccac 20 50 20 DNA ArtificialSequence Antisense Oligonucleotide 50 ttgatttctt ctgtttcagc 20 51 20 DNAArtificial Sequence Antisense Oligonucleotide 51 tggctgagaa tacgagctgg20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 gtccactgagtttcattcta 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53gtgaaagtaa agatagtttt 20 54 20 DNA Artificial Sequence AntisenseOligonucleotide 54 aaactgtgga gtgaaagtaa 20 55 20 DNA ArtificialSequence Antisense Oligonucleotide 55 tagaactgtt gctggtctat 20 56 20 DNAArtificial Sequence Antisense Oligonucleotide 56 tccagagcca ggtagaactg20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 cagcggctacagaggtagga 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58ggtgggctgg cctgtcatcc 20 59 20 DNA Artificial Sequence AntisenseOligonucleotide 59 ttccatcttc atcaagcatg 20 60 20 DNA ArtificialSequence Antisense Oligonucleotide 60 aggatacttg aattcaagct 20 61 20 DNAArtificial Sequence Antisense Oligonucleotide 61 catttttcgg agtgctgcca20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 tctgacaatttacctgtttg 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63tgttgtcaaa aactctgaca 20 64 20 DNA Artificial Sequence AntisenseOligonucleotide 64 caagatgttg tcaaaaactc 20 65 20 DNA ArtificialSequence Antisense Oligonucleotide 65 ggtccagggt ccatgaagct 20 66 20 DNAArtificial Sequence Antisense Oligonucleotide 66 gcttaccctc aggtccaggg20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 atcataatcttcactgtaca 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68ttcatcacca cagcgagcat 20 69 20 DNA Artificial Sequence AntisenseOligonucleotide 69 accgatctag ccctcccttc 20 70 20 DNA ArtificialSequence Antisense Oligonucleotide 70 accaaggaca ttaagtcgca 20 71 20 DNAArtificial Sequence Antisense Oligonucleotide 71 atacatgtga acattctgta20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 ttgaggtctccttcaactgt 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73ttccagtcag gtcctttcat 20 74 20 DNA Artificial Sequence AntisenseOligonucleotide 74 gtgcaggcct catcaagtgc 20 75 20 DNA ArtificialSequence Antisense Oligonucleotide 75 tggaggaagt cctactgtca 20 76 20 DNAArtificial Sequence Antisense Oligonucleotide 76 actggcttca tctatcagct20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 ccattatttcctgtgtaagg 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78tgccataact tgtatgatca 20 79 20 DNA Artificial Sequence AntisenseOligonucleotide 79 tctgtggctt cctcagctga 20 80 20 DNA ArtificialSequence Antisense Oligonucleotide 80 gactgagatt catcaggccc 20 81 20 DNAArtificial Sequence Antisense Oligonucleotide 81 ctgagaatgt gatgcaggag20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 cttcgttccactccaaactc 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83ttgaatcagt gggacgaacg 20 84 20 DNA Artificial Sequence AntisenseOligonucleotide 84 gctccaacag caccaatctc 20 85 20 DNA ArtificialSequence Antisense Oligonucleotide 85 tcacttttta actgcatgat 20 86 20 DNAArtificial Sequence Antisense Oligonucleotide 86 gaaattccac ctgctttatc20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 ttgacgactatctttagatg 20 88 20 DNA Artificial Sequence Antisense Oligonucleotide 88gagaatttaa tctcacctgg 20 89 20 DNA Artificial Sequence AntisenseOligonucleotide 89 ttcaggacag actccacatg 20 90 20 DNA ArtificialSequence Antisense Oligonucleotide 90 acaaggatgg cttcaaccag 20 91 20 DNAArtificial Sequence Antisense Oligonucleotide 91 atatctgcca gcatggtgag20 92 20 DNA Artificial Sequence Antisense Oligonucleotide 92 atgaatttcaatatctgcca 20 93 20 DNA Artificial Sequence Antisense Oligonucleotide 93tgttcttgaa ggaacaagtc 20 94 20 DNA Artificial Sequence AntisenseOligonucleotide 94 aacatggtat catctgcgcc 20 95 20 DNA ArtificialSequence Antisense Oligonucleotide 95 atcctttgcc aacatggtat 20 96 20 DNAArtificial Sequence Antisense Oligonucleotide 96 agagtacaga tgccagatgc20 97 20 DNA Artificial Sequence Antisense Oligonucleotide 97 gccactgggtgcactgtcat 20 98 20 DNA Artificial Sequence Antisense Oligonucleotide 98aggtaggtca tggtgccaaa 20 99 20 DNA Artificial Sequence AntisenseOligonucleotide 99 aactcctgca cgtaggtggc 20 100 20 DNA ArtificialSequence Antisense Oligonucleotide 100 ccaaagccct cattgcatgg 20 101 20DNA Artificial Sequence Antisense Oligonucleotide 101 gtccaagctatgacaagcta 20 102 20 DNA Artificial Sequence Antisense Oligonucleotide102 ataatgggta ttattaaaag 20 103 20 DNA Artificial Sequence AntisenseOligonucleotide 103 ccggctgcag aggtaggaga 20 104 20 DNA ArtificialSequence Antisense Oligonucleotide 104 tccaccggct gcagaggtag 20 105 20DNA Artificial Sequence Antisense Oligonucleotide 105 tcctccaccggctgcagagg 20 106 20 DNA Artificial Sequence Antisense Oligonucleotide106 gtcatcctcc accggctgca 20 107 20 DNA Artificial Sequence AntisenseOligonucleotide 107 gggctggcct gtcatcctcc 20 108 20 DNA ArtificialSequence Antisense Oligonucleotide 108 agtgatggtg ggctggcctg 20 109 20DNA Artificial Sequence Antisense Oligonucleotide 109 aaagtgatggtgggctggcc 20 110 20 DNA Artificial Sequence Antisense Oligonucleotide110 agcatggtgt gtgagatggg 20 111 20 DNA Artificial Sequence AntisenseOligonucleotide 111 cagcatggtg tgtgagatgg 20 112 20 DNA ArtificialSequence Antisense Oligonucleotide 112 catccagcat ggtgtgtgag 20 113 20DNA Artificial Sequence Antisense Oligonucleotide 113 cttcatccagcatggtgtgt 20 114 20 DNA Artificial Sequence Antisense Oligonucleotide114 tcttcatcca gcatggtgtg 20 115 20 DNA Artificial Sequence AntisenseOligonucleotide 115 tccatcttca tccagcatgg 20 116 20 DNA ArtificialSequence Antisense Oligonucleotide 116 ggctccatct tcatccagca 20 117 20DNA Artificial Sequence Antisense Oligonucleotide 117 gctggctccatcttcatcca 20 118 20 DNA Artificial Sequence Antisense Oligonucleotide118 caagctggct ccatcttcat 20 119 20 DNA Artificial Sequence AntisenseOligonucleotide 119 ttgaattcaa gctggctcca 20 120 20 DNA ArtificialSequence Antisense Oligonucleotide 120 ggatacttga attcaagctg 20 121 20DNA Artificial Sequence Antisense Oligonucleotide 121 caggatacttgaattcaagc 20 122 20 DNA Artificial Sequence Antisense Oligonucleotide122 ccaggatact tgaattcaag 20 123 20 DNA Artificial Sequence AntisenseOligonucleotide 123 tgccaggata cttgaattca 20 124 20 DNA ArtificialSequence Antisense Oligonucleotide 124 ctgccaggat acttgaattc 20 125 20DNA Artificial Sequence Antisense Oligonucleotide 125 gctgccaggatacttgaatt 20 126 20 DNA Artificial Sequence Antisense Oligonucleotide126 tgctgccagg atacttgaat 20 127 20 DNA Artificial Sequence AntisenseOligonucleotide 127 gagtgctgcc aggatacttg 20 128 20 DNA ArtificialSequence Antisense Oligonucleotide 128 ggagtgctgc caggatactt 20 129 20DNA Artificial Sequence Antisense Oligonucleotide 129 tccggagtgctgccaggata 20 130 20 DNA Artificial Sequence Antisense Oligonucleotide130 ttttccggag tgctgccagg 20 131 20 DNA Artificial Sequence AntisenseOligonucleotide 131 cattttccgg agtgctgcca 20 132 20 DNA ArtificialSequence Antisense Oligonucleotide 132 gcattttccg gagtgctgcc 20 133 20DNA Artificial Sequence Antisense Oligonucleotide 133 cctgcattttccggagtgct 20 134 20 DNA Artificial Sequence Antisense Oligonucleotide134 catcctgcat tttccggagt 20 135 20 DNA Artificial Sequence AntisenseOligonucleotide 135 aaccatcctg cattttccgg 20 136 20 DNA ArtificialSequence Antisense Oligonucleotide 136 aataaccatc ctgcattttc 20 137 20DNA Artificial Sequence Antisense Oligonucleotide 137 caaaataaccatcctgcatt 20 138 20 DNA Artificial Sequence Antisense Oligonucleotide138 accaaaataa ccatcctgca 20 139 20 DNA Artificial Sequence AntisenseOligonucleotide 139 ccaccaaaat aaccatcctg 20 140 20 DNA ArtificialSequence Antisense Oligonucleotide 140 ggccccacca aaataaccat 20 141 20DNA Artificial Sequence Antisense Oligonucleotide 141 atcctggccccaccaaaata 20 142 20 DNA Artificial Sequence Antisense Oligonucleotide142 tttggatcct ggccccacca 20 143 20 DNA Artificial Sequence AntisenseOligonucleotide 143 cctgtttgga tcctggcccc 20 144 20 DNA ArtificialSequence Antisense Oligonucleotide 144 ttacctgttt ggatcctggc 20 145 20DNA Artificial Sequence Antisense Oligonucleotide 145 agcttacctgtttggatcct 20 146 20 DNA Artificial Sequence Antisense Oligonucleotide146 gacagcttac ctgtttggat 20 147 20 DNA Artificial Sequence AntisenseOligonucleotide 147 tctgacagct tacctgtttg 20 148 20 DNA ArtificialSequence Antisense Oligonucleotide 148 aactctgaca gcttacctgt 20 149 20DNA Artificial Sequence Antisense Oligonucleotide 149 aaaaactctgacagcttacc 20 150 20 DNA Artificial Sequence Antisense Oligonucleotide150 gtcaaaaact ctgacagctt 20 151 20 DNA Artificial Sequence AntisenseOligonucleotide 151 gttgtcaaaa actctgacag 20 152 20 DNA ArtificialSequence Antisense Oligonucleotide 152 gatgttgtca aaaactctga 20 153 20DNA Artificial Sequence Antisense Oligonucleotide 153 cagcaagatgttgtcaaaaa 20 154 20 DNA Artificial Sequence Antisense Oligonucleotide154 gtgcagcaag atgttgtcaa 20 155 20 DNA Artificial Sequence AntisenseOligonucleotide 155 tgtgtgcagc aagatgttgt 20 156 20 DNA ArtificialSequence Antisense Oligonucleotide 156 tgaagcttaa gtgtgtgcag 20 157 20DNA Artificial Sequence Antisense Oligonucleotide 157 gggtccatgaagcttaagtg 20 158 20 DNA Artificial Sequence Antisense Oligonucleotide158 tccagggtcc atgaagctta 20 159 20 DNA Artificial Sequence AntisenseOligonucleotide 159 taatcttcgc tgtacagctt 20 160 20 DNA ArtificialSequence Antisense Oligonucleotide 160 gtcttcatca taatcttcgc 20 161 20DNA Artificial Sequence Antisense Oligonucleotide 161 tagtcttcatcataatcttc 20 162 20 DNA Artificial Sequence Antisense Oligonucleotide162 tcatagtctt catcataatc 20 163 20 DNA Artificial Sequence AntisenseOligonucleotide 163 tcatcatcat agtcttcatc 20 164 20 DNA ArtificialSequence Antisense Oligonucleotide 164 ccagctcatc atcatagtct 20 165 20DNA Artificial Sequence Antisense Oligonucleotide 165 gtccagctcatcatcatagt 20 166 20 DNA Artificial Sequence Antisense Oligonucleotide166 agtccagctc atcatcatag 20 167 20 DNA Artificial Sequence AntisenseOligonucleotide 167 agagtccagc tcatcatcat 20 168 20 DNA ArtificialSequence Antisense Oligonucleotide 168 tgccagagtc cagctcatca 20 169 20DNA Artificial Sequence Antisense Oligonucleotide 169 atccagttgccagagtccag 20 170 20 DNA Artificial Sequence Antisense Oligonucleotide170 catccagttg ccagagtcca 20 171 20 DNA Artificial Sequence AntisenseOligonucleotide 171 atccatccag ttgccagagt 20 172 20 DNA ArtificialSequence Antisense Oligonucleotide 172 gctatccatc cagttgccag 20 173 20DNA Artificial Sequence Antisense Oligonucleotide 173 catagctatccatccagttg 20 174 20 DNA Artificial Sequence Antisense Oligonucleotide174 gagtcatagc tatccatcca 20 175 20 DNA Artificial Sequence AntisenseOligonucleotide 175 cgtgttgagt catagctatc 20 176 20 DNA ArtificialSequence Antisense Oligonucleotide 176 acgtgttgag tcatagctat 20 177 20DNA Artificial Sequence Antisense Oligonucleotide 177 cattacgtgttgagtcatag 20 178 20 DNA Artificial Sequence Antisense Oligonucleotide178 gcattacgtg ttgagtcata 20

What is claimed is:
 1. A antisense compound 8 to 50 nucleobases inlength targeted to a start codon region, a coding region, a stop codonregion, or a 3′-untranslated region of human Phosphorylase kinase alpha1 (SEQ ID NO: 3) or a coding region of mouse Phosphorylase kinase alpha1 (SEQ ID NO: 10) or a coding region of rat Phosphorylase kinase alpha 1(SEQ ID NO: 17), wherein said antisense compound specifically hybridizeswith one of said regions and inhibits the expression of Phosphorylasekinase alpha
 1. 2. The compound of claim 1 which is an antisenseoligonucleotide.
 3. The compound of claim 2 wherein the antisenseoligonucleotide comprises at least one modified internucleoside linkage.4. The compound of claim 3 wherein the modified internucleoside linkageis a phosphorothioate linkage.
 5. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modified sugar moiety.6. The compound of claim 5 wherein the modified sugar moiety is a2′-O-methoxyethyl sugar moiety.
 7. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modified nucleobase. 8.The compound of claim 7 wherein the modified nucleobase is a5-methylcytosine.
 9. The compound of claim 2 wherein the antisenseoligonucleotide is a chimeric oligonucleotide.
 10. A compositioncomprising the compound of claim 1 and a pharmaceutically acceptablecarrier or diluent.
 11. The composition of claim 10 further comprising acolloidal dispersion system.
 12. The composition of claim 10 wherein thecompound is an antisense oligonucleotide.
 13. A method of inhibiting theexpression of Phosphorylase kinase alpha 1 in cells or tissuescomprising contacting said cells or tissues in vitro with the antisensecompound of claim 1 so that expression of Phosphorylase kinase alpha 1is inhibited.
 14. A compound up to 50 nucleobases in length comprisingat least an 8-nucleobase portion of SEQ ID NO: 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 103, 104, 105, 106,107, 108, 109, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 or178 which inhibits the expression of Phosphorylase kinase alpha
 1. 15.The antisense compound of claim 14 which is an antisenseoligonucleotide.
 16. The antisense compound of claim 15 wherein theantisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 17. The antisense compound of claim 16 whereinthe modified internucleoside linkage is a phosphorothioate linkage. 18.The antisense compound of claim 15 wherein the antisense oligonucleotidecomprises at least one modified sugar moiety.
 19. The antisense compoundof claim 18 wherein the modified sugar moiety is a 2′-O-methoxyethylsugar moiety.
 20. The antisense compound of claim 15 wherein theantisense oligonucleotide comprises at least one modified nucleobase.21. The antisense compound of claim 20 wherein the modified nucleobaseis a 5-methylcytosine.
 22. The antisense compound of claim 15 whereinthe antisense oligonucleotide is a chimeric oligonucleotide.
 23. Amethod of inhibiting the expression of Phosphorylase kinase alpha 1 incells or tissues comprising contacting said cells or tissues in vitrowith the antisense compound of claim 14 so that expression ofPhosphorylase kinase alpha 1 is inhibited.
 24. A composition comprisingthe antisense compound of claim 14 and a pharmaceutically acceptablecarrier or diluent.
 25. The composition of claim 24 further comprising acolloidal dispersion system.
 26. The composition of claim 24 wherein theantisense compound is an antisense oligonucleotide.